Figure 7.

Fusion can occur without the granule occurring in the evanescent field. Three events are shown (9, 173, and 209) in which fusion occurs with the granule not evident in the evanescent field within 100 ms of the event. For comparison, event 13 (top sequence) shows a granule in the evanescent field immediately before fusion. Images were taken at 10 Hz. Zero time is the frame just before the fusion event that is characterized by spreading and increase in the intensity. The arrows indicate the position of the fusion event and are identically placed in the frames within a sequence. The maximum postfusion intensity (in arbitrary units; a.u.) reflects the total amount of VAMP-GFP in the granule after it is exposed to the extracellular medium at pH 7.4. The median postfusion fluorescence of granules that were present in the evanescent field before fusion was 2000 a.u. Note that event 9 shows a hint of an out-of-focus granule at -0.1 and 0 s. The out-of-focus granule is probably visualized because of a small amount of contaminating propagated light caused by spurious reflections in the objective lens (Mattheyses and Axelrod, 2006) and/or interaction of the evanescent field with highly refractive intracellular structures (Oheim and Stuhmer, 2000). Bar, 1 μm.